Study of antigenic structure and inhibition of activity of human growth hormone and chorionic somatomammotropin by monoclonal antibodies

Distinct antigenic determinants were identified on native molecules of human growth hormone (hGH) and chorionic somatomammotropin (hCS) on the basis of competitive inhibition assays with eight murine monoclonal antibodies. Effective competition for antigen binding within a pair of antibodies indicated overlapping combining site specificities whereas a lack of competition suggested binding to sterically distinct structural moieties. An antigenic determinant, specific for hGH was detected by antibodies QA68 and NA27. whilst another marginally hCS-cross-reactive site was bound by NA71. Two distinct determinants fully expressed by either hGH or hCS were bound by antibody pairs NA39/EB2 and EBI/EB3 respectively, whereas a single hCS-specific determinant was recognized by antibody EB4. An unexpected reciprocal cross-inhibition of soluble antigen-antibody complex binding was observed between antibodies reacting to distinct determinants, i.e. for NA27 towards NA39/EB2 and for NA71 towards EBI/EB3. These results were tentatively interpreted in terms of conformational changes of antigen when bound in soluble immune complexes, but an alternative explanation of steric hindrance cannot yet be excluded. The effect of monoclonal antibodies on the hormonal biological activity was investigated in a dose-response study of the hormone-dependent growth stimulation of NB2 lymphoma cells in tissue culture. Although all eight antibodies were specifically growth-inhibitory, major quantitative differences in their efficacies have been observed. At limiting hormone doses antibodies EB2/NA39 were most effective whereas QA68 and NA71 were the most potent at excess hormone input. Various mechanisms operating through inhibition of hormone binding and/or modulation of cell receptor-bound complexes have been considered.     

Detection of circulating immune complexes with a Raji cell enzyme immunoassay

The primary aim of this work was to produce specific monoclonal antibodies to human growth hormone (hGH) for use in a diagnostic RIA of hGH levels in serum. Three different schedules were used for immunization of BALB/c mice and the splenocytes from each mouse were fused with myeloma cells Sp 2/0 Ag 14. Each fusion resulted in the production of hundreds of hybridomas secreting hGH-directed antibodies. Six antibodies have been fully characterized and have been grouped into pairs which recognize 3 different epitopes on the hGH molecule. One pair exhibits no cross reaction with the structurally related placental hormone, human placental lactogen (hPL), a second pair has low cross reaction with hPL (1.6-3%) and a third pair reacts equally well with hGH and hPL indicating binding to a common epitope in the 2 molecules. The highest affinity antibody, 74/6, which has an affinity constant of 4.4 X 10(10) l/mol and 3% cross-reactivity with hPL, has been used to establish a RIA for serum hGH measurements. Evidence is provided that hGH levels measured in this assay correlate well with those obtained in a conventional rabbit antiserum assay.     

Comparative study of pituitary and bacteria-derived human growth hormone by monoclonal antibodies

We have established hybridoma lines which secrete mouse monoclonal antibodies (Mabs) to human pituitary growth hormone, hGH. Using indirect competitive ELISA and indirect passive hemagglutination inhibition twelve different Mabs were characterized with regard to cross-reactivity with the hGH-related hormones, human chorionic somatomammotropin, hCS, and human prolactin, hPRL. The reactivity of these Mabs with pituitary hGH was compared to that with either bacterially-produced methionyl-hGH or to that of reduced and S-carboxymethylated hGH, which has an altered conformation. None of the Mabs reacted with hPRL. Four did not react with hCS whereas the others showed varying degree of cross-reactivity with hCS. All Mabs reacted more weakly with reduced and S-carboxymethylated hGH than with the native form of the hormone, which was not seen with conventional rabbit antisera to hGH. Thus in the case of hGH the Mabs are superior to conventional antisera in revealing small conformational differences. However the pituitary and bacterially-derived methionyl-hGH were indistinguishable as determined by the 12 Mabs.     

Studies of the low dose 'hook' effect in a competitive homogeneous immunoassay.

The interactions of two monoclonal antibodies with human growth hormone (hGH) have been investigated. The individual antibodies showed normal behavior in a competitive binding assay, but mixtures of the antibodies demonstrated a 'hook' attributable to cooperative interactions. Cooperativity was observed in titrations which preceded the competitive binding assay. Size exclusion chromatographic data suggest that the cooperativity is explained by the formation of higher molecular weight complexes (up to 700 kDa). The major complex is probably linear, consisting of three antibody molecules. Circular and linear complexes with four antibody molecules (octameric complexes) are also possible. Theoretical models also support the formation of cyclic complexes in a competitive binding assay.     

Binding characteristics of monoclonal antibodies raised against bovine growth hormone

Monoclonal antibodies have been raised against pituitary bovine growth hormone using the hybridoma procedure. The binding characteristics of the seven selected monoclonal antibodies toward the antigen molecule in its native, chemically or enzymatically treated form have been studied. The reactivities of the monoclonal antibodies with growth hormones from other species and bovine prolactin have also been investigated. The epitopes recognized by four of the produced monoclonal antibodies are conformational, whereas two other monoclonal antibodies bind to sequential determinants. Three antibodies define immunological sites located between residues 6-124 of the bovine growth hormone molecule, and one of this antibody shows higher affinity to human than bovine growth hormone. The immunoreactivity of one monoclonal antibody is enhanced by the previous binding of the antigen to polyclonal antibodies, probably because of a localized conformational change of the bovine growth hormone molecule. This antibody also shows cross-reactivity with all the homologous hormones tested, indicating to recognize a highly conserved antigenic determinant.     

The recognition by monoclonal antibodies of various portions of a major antigenic site of human growth hormone

The reactivities of five mouse monoclonal antibodies against human growth hormone (hGH) were defined by either a competitive radioimmunoassay with insolubilized antibodies or by an agglutination-inhibition method with hGH-coated polystyrene particles. The five antibodies reacted significantly but to various degrees with human placental lactogen and at least three antibodies reacted with human prolactin and three synthetic peptides extending from residues 19 to 128, 73 to 128 and 98 and 128 of hGH. Four tested monoclonal antibodies failed to react with bovine growth hormone and with hGH oxidized by performic acid. The antibodies were further distinguished by their different reactions with hGH modified by reduction and alkylation or by adsorption on a polystyrene surface. The unique specificity of each antibody was confirmed for most of them by an agglutination method in which the agglutinating activity of hGH was tested on latex particles coated with various paired combinations of the monoclonal antibodies. The lack of agglutination with certain combinations suggested that the specificities of such a pair of antibodies overlapped each other. These results suggest that the sequences corresponding to the synthetic peptides participate in the structure of a major antigenic site of which various portions are recognized by the monoclonal antibodies.     

Monoclonal antibodies to human growth hormone induce an allosteric conformational change in the antigen.

We re-investigated the properties of a monoclonal antibody (mAb), 4D11, to human growth hormone (hGH) that showed a very weak affinity, recognizing hGH only when the hormone was solubilized on a solid surface. MAb4D11 did not significantly bind 125I-hGH. It was found that three mAb directed to different hGH epitopes (mAb 3C11, 10C1 and NA71) were able to induce the binding of the soluble antigen to mAb 4D11. The co-operative effect could be demonstrated by the formation of binary complexes (Ag:Ab, 1:2) detected by high-performance liquid chromatography (HPLC) and by the increase of radioactivity found when the synergistic mAb were added to 125I-hGH incubated with mAb 4D11 immobilized on polyvinyl microplates. Other possible explanations, such as the formation of cyclic complexes or the generation of a new epitope in the Fc fragment of the first antibody (Ab), were dismissed because the Fab fragment of one of the enhancing mAb (3C11) gave the same effect as the intact Ab. The data suggest that the hGH molecule undergoes a localized conformational change after binding to mAb 3C11, NA71 or 10C1 and that mAb 4D11 binds with high affinity to the modified region of the hormone. The formation or not of ternary complexes (Ag:Ab, 1:3) was used to localize the 4D11 epitope on the surface of the Ag. It is suggested that mAb 4D11 recognizes a conformational change produced in the region defined by the AE5/AC8 epitopes, which is close to the hGH antigenic domain only expressed when the protein is immobilized on plastic surfaces.     

Two-site monoclonal antibody quantitative ELISA for toxic shock syndrome toxin-1

A two-site monoclonal antibody (MAB) quantitative enzyme-linked immunosorbent assay (ELISA) was developed that enables quantitation of toxic shock syndrome toxin-1 (TSST-1) down to 0.25 ng/ml and detection of TSST-1 to 0.06 ng/ml. Interference by Staphylococcus protein A was eliminated by incorporating normal rabbit serum into the test sample diluent. In the process of selecting an MAB pair for a two-site 'sandwich'-type ELISA, the MABs were screened for inhibition or common epitope binding. Some MABs that reacted with antigen that was adsorbed to a polystyrene well would not bind to antigen that was presented in a more natural configuration, as in the case of antigen immobilized by trapping antibody. Conversely, MABs that reacted with antigen that was immobilized by another antibody did not all function as trapping antibodies when adsorbed directly to a polystyrene surface. ELISAs that used polyclonal antibodies in the capture mode and MAB conjugate as the second antibody were generally more sensitive than were those that used polyclonal antibodies for both capture and indicator functions. MAB screening and selection schemes should be carefully designed to evaluate MABs in the mode in which they will be used in the final assay.     

Enhancement of bovine growth hormone activity in vivo by monoclonal antibodies

Monoclonal antibodies (MAbs) prepared against ovine growth hormone (oGH) and recognizing several distinct or related specificities on bovine growth hormone (bGH), have been shown to strikingly enhance the growth promoting properties of the hormone in vivo as determined by 35SO2-4 incorporation in cartilage. The phenomenon is dependent on both hormone and antibody dose and is saturable in the presence of excess antibody. Competition binding analysis between pairs of antibodies has shown that they define distinct antigenic regions on bGH of which one is represented by four MAbs. The most potent growth enhancement was associated with a group of three MAbs (OA11, OA12 and OA13) binding to topographically closely related sites. Another MAb (OA14) with a specificity similar to OA11 and OA13 as defined by competition assay failed to enhance bGH activity. Two antibodies binding to a further two sites (OA15 and OA16) demonstrated modest growth enhancement activity when in complex with bGH, whereas the binding of another antibody (OA17) did not significantly affect hormonal activity. Univalent antibody fragments (Fab) derived from MAb OA11 were equi-potent to the bivalent form of the antibody in the enhancement of bGH activity. Determination of the effects of the different MAbs on the binding of 125I-bGH to liver microsome receptors revealed substantial increase in specific binding (3.5-fold) and is associated with some growth enhancing MAbs. However, not all growth enhancing MAbs increased receptor binding and in the case of one, OA16, clear cut inhibition of receptor binding was observed. Tentative conclusions have been drawn on the possible underlying mechanisms of the MAb mediated enhancement phenomenon.     

Multiple epitope interactions in the two-step sandwich immunoassay.

The 'hook' effect as related to the two-step sandwich immunoassay has been investigated experimentally and theoretically. The multiple epitope interactions between the analyte and the labeled antibody cause a 'hook' in the two-step sandwich immunoassay. Three different analytes and monoclonal antibodies were chosen to carefully demonstrate the effect of the analyte characteristics on this immunoassay. Two monoclonal antibodies against two different epitopes of biosynthetic human growth hormone (hGH) was the simplest model for this study. The sandwich immunoassay for hGH shows no 'hook' effect. The non-covalent dimeric form of hGH (D-hGH) possesses two repeating epitopes which is the simplest model for an analyte having a discrete number of repeating epitopes. The D-hGH assay demonstrated a 'hook' effect in the two-step sandwich immunoassay if the labeled antibody was allowed to interact with more than one epitope. In a third system multiple epitope interactions with the labeled antibody were observed using ferritin. The effect of the analyte concentration and the liquid-phase antibody have been examined to elucidate the nature of these various interactions. The cause of the 'hook' effect in the two-step sandwich immunoassay is attributed to the desorption of the bound analyte due to a conformational change after the labeled antibody interacts with several epitopes of the adsorbed analyte.     

The production and characterisation of monoclonal antibodies against human prolactin and the development of a two-site immunoradiometric assay

Monoclonal antibodies against human prolactin (PRL) have been produced and characterised and used to develop a sensitive two-site immunoradiometric assay (IRMA). Nine anti-PRL monoclonal antibodies were assessed for reactivity in immunoblotting experiments with PRL, hPL, hGH and pituitary gland extract. There was no detectable crossreactivity with hPL or hGH. In liquid phase radioimmunoassay (RIA) studies using three of the antibodies there was no detectable crossreaction from hPL or hGH. Five antibodies were positive in immunocytochemical studies using sections of human pituitary gland. Using FPLC purified monoclonal antibodies, a two-site IRMA was developed that could assay PRL over the range 17.5-3500 mIU per litre and was readily adapted to assaying serum samples from patients. The two-site IRMA could be performed within one day without loss of sensitivity and has potential as a rapid and simple method for screening clinical samples.     

ELISA for Determination of Human Growth Hormone: Recognition of Helix 4 Epitopes

Human growth hormone (hGH) signal transduction initiates with areceptor dimerization in which one molecule binds to the receptorthrough sites 1 and 2. A sandwich enzyme-linked immunosorbentassay was developed for quantifying hGH molecules that presenthelix 4 from binding site 1. For this, horse anti-rhGH antibodieswere eluted by an immunoaffinity column constituted bysepharose-rhGH. These antibodies were purified through a secondcolumn with synthetic peptide correspondent to hGH helix4, immobilized to sepharose, and used as capture antibodies.Those that did not recognize synthetic peptide were used as amarker antibody. The working range was of 1.95 to 31.25ng/mLof hGH. The intra-assay coefficient of variation (CV) was between4.53% and 6.33%, while the interassay CV was between 6.00% and8.27%. The recovery range was between 96.0% to 103.8%. Therewas no cross-reactivity with human prolactin. These features showthat our assay is an efficient method for the determination of hGH.     

Monoclonal antibodies to human chorionic gonadotropin and their application to two-site sandwich radioimmunoassay

Monoclonal antibodies were prepared against human chorionic gonadotropin (HCG). One monoclonal antibody recognized a conformational determinant expressed only on native HCG molecule and another monoclonal antibody had the specificity for the epitopes located on the beta-subunit of HCG. Monoclonal antibodies reacting with different antigenic determinants on the HCG molecule were used to develop a simplified 2-site sandwich radioimmunoassay in which one monoclonal antibody was immobilized and another labeled with 125iodine. This assay was highly specific for HCG and there was no cross-reactivity with alpha, beta-subunit of HCG, luteinizing hormone and follicle stimulating hormone.     

Production and characterization of monoclonal antibodies against Clostridium perfringens type A enterotoxin.

Hybridomas secreting monoclonal antibodies (MABs) specific for Clostridium perfringens type A enterotoxin were produced by fusion of P3X63Ag8.653 myeloma cells with spleen cells from BALB/c mice immunized with purified enterotoxin. Wells containing hybridomas secreting immunoglobulin G (IgG) antibodies against enterotoxin were specifically identified by an indirect enzyme-linked immunosorbent assay (ELISA), and 10 ELISA-positive hybridomas were selected and cloned twice by limiting dilution. All 10 hybridomas produced MABs containing immunoglobulin G1 heavy chains and kappa (kappa) light chains. These hybridomas were then grown as ascitic tumors in mice, and MABs were purified from the ascites fluids with DEAE Affi-gel blue. The specificity of the MABs for enterotoxin was demonstrated by immunoblotting and ELISA. Competitive radioimmunoassay with 125I-MABs suggests that these MABs recognized at least four epitopes on the enterotoxin molecule. The enterotoxin-neutralizing ability of MABs from both hybridoma culture supernatants and ascites fluids was assessed by using a 3H-nucleotide-release Vero (African green monkey kidney) cell assay. Only 2 of the 10 hybridomas produced MABs which completely (greater than 90%) neutralized the biologic activity of enterotoxin. Preincubation of 125I-enterotoxin with MABs demonstrated that MAB neutralizing ability correlated with MAB-specific inhibition of specific binding of enterotoxin to intestinal brush border membranes.     

Production and characterization of monoclonal antibodies to human growth hormone

Three BIOZZI-HR mice were immunized with human growth hormone (hGH). From the determination of the titer, the average equilibrium association constant and the heterogeneity index of the antisera, it was possible to select the most suitable mouse for production of monoclonal antibodies (Mabs). Resulting from a single fusion, eight Mabs were produced, purified and characterized. The equilibrium association constant of the Mabs ranged from 5.10(8) M-1 to 9.109 M-1 at physiological pH. Four areas on hGH are recognized by the Mabs (the topology of the Mabs was investigated by two-site immunoradiometric assays). The Mabs, which recognize a same area, show similar cross-reactivities between hGH and human Placental Lactogen (hPL). No selected Mabs bound human Prolactin (hPRL), equine Growth Hormone (eGH) and porcine Growth Hormone (pGH). Two complementary Mabs enable a two-site immunometric assay of pituitary and E. Coli derived hGH.     

人生长激素基因真核表达载体的构建及鉴定

目的 构建人生长激素的真核细胞表达载体,测定并分析目的 基因序列.方法 用PCR方法扩增出人生长激素基因,连接至T克隆载体,然后分别亚克隆至真核细胞表达载体pCDNA3.1、pCEP4中.使用DNA ssist软件分析序列.结果 所构建真核表达载体的酶切鉴定、PCR鉴定、测序鉴定均正确.结论 成功构建出多个人生长激素基因真核细胞表达载体,验证了基因序列的正确性,为下一步基因治疗研究打下基础.     

Studies of the 'hook' effect in the one-step sandwich immunoassay.

The one-step sandwich immunoassay is increasingly replacing the traditional two-step immunoassay due to obvious advantages such as assay speed. However, the one-step sandwich immunoassay suffers from the 'hook' effect irrespective of the analyte characteristics. The 'hook' effect is dependent primarily on the analyte concentration. Three different model analytes, human growth hormone (hGH), the dimeric form of hGH (D-hGH, having a discrete number of repeating epitopes) and ferritin (multiple epitopes) having different immunological properties have been employed in studies of the one-step sandwich immunoassay. The characteristics of each of the model analytes offer new insights into general guidelines for assay procedures. These guidelines permit rapid optimization of assay conditions for an immunoassay without a priori knowledge of the immunological characteristics of the antibody or antigen. Both experimental and theoretical data show several instances where high capacity solid-phase antibodies can effectively shift the 'hook' to relatively higher analyte concentrations. The effect of the concentration of labeled antibody on assay response was examined theoretically.     

Transgenesis and neuroendocrine physiology: a transgenic rat model expressing growth hormone in vasopressin neurones

Human growth hormone (hGH) and bovine neurophysin (bNP) DNA reporter fragments were inserted into the rat vasopressin (VP) and oxytocin (OT) genes in a 44 kb cosmid construct used to generate two lines of transgenic rats, termed JP17 and JP59. Both lines showed specific hGH expression in magnocellular VP cells in the hypothalamic paraventricular (PVN) and supraoptic nuclei (SON). hGH was also expressed in parvocellular neurones in suprachiasmatic nuclei (SCN), medial amygdala and habenular nuclei in JP17 rats; the rat OT-bNP (rOT-bNP) transgene was not expressed in either line. Immunohistochemistry and radioimmunoassay showed hGH protein in the hypothalamus from where it was transported in varicose fibres via the median eminence to the posterior pituitary gland. Immunogold electron microscopy showed hGH co-stored with VP-NP in the same granules. The VP-hGH transgene did not affect water balance, VP storage or release in vivo . Drinking 2 % saline for 72 h increased hypothalamic transgene hGH mRNA expression, and depleted posterior pituitary hGH and VP stores in parallel. In anaesthetised, water-loaded JP17 rats, hGH was released with VP in response to an acute hypovolumic stimulus (sodium nitrosopentacyano, 400 μg I.V.). JP17 rats had a reduced growth rate, lower anterior pituitary rGH contents, and a reduced amplitude of endogenous pulsatile rGH secretion assessed by automated blood microsampling in conscious rats, consistent with a short-loop feedback of the VP-hGH on the endogenous GH axis. This transgenic rat model enables us to study physiological regulation of hypothalamic transgene protein production, transport and secretion, as well as its effects on other neuroendocrine systems in vivo .     

Recognition of HLA-B27 and related antigen by a monoclonal antibody

A monoclonal antibody that binds specifically to HLA-B27, B7, and B22 is described. Binding to B27 appeared to be slightly stronger than to B7 and stronger than to B22 in an indirect binding assay, but no difference in B7 and B27 binding could be detected by Scatchard analysis. No distinction could be made between B27 on cells from normal and from ankylosing spondylitis patients in any assay system. The antibody, which was not cytotoxic, blocked complement-dependent cytolysis mediated by human HLA typing sera specific for B7 and B27. Competitive binding studies with other monoclonal antibodies showed that ME1 could block the binding of antibodies that recognized different antigenic sites on HLA. ME1 did not bind to Klebsiella pneumoniae. This reagent will be useful in further analysis of the relationship between B27 and ankylosing spondylitis.     

Human leukocytic pyrogen test for detection of pyrogenic material in growth hormone produced by recombinant Escherichia coli.

Human growth hormone is biosynthetically produced in recombinant strains of Escherichia coli as methionyl human growth hormone (met-hGH). When purified from the bacterial culture, met-hGH is biologically active in established assays for growth hormone. Therefore, a phase I trial of met-hGH was carried out in healthy human adults; during the first trial, however, signs, symptoms, and clinical laboratory tests characteristic of an acute-phase response to pyrogenic agents was observed. Prior testing of the met-hGH preparation used in the phase I trial did not reveal evidence of toxicity, and the U.S. Pharmacopeial Convention rabbit pyrogen test, as well as the Limulus amoebocyte lysate (LAL) test, had not detected significant levels of exogenous pyrogens or endotoxin. In addition, standard inhibition studies with added endotoxin showed no inhibition by the LAL test. When this preparation of met-hGH was incubated with human blood mononuclear cells, leukocytic pyrogen (LP) was released into the supernatant medium, suggesting that the preparation contained pyrogenic material. Various lots of met-hGH based on different purification and formulating methods were tested by the human LP assay for contaminating pyrogens. The results of these tests aided in the identification of procedures for met-hGH preparations which did not induce LP in vitro. Thus, subsequent lots of met-hGH which had passed the LP test were used in repeat clinical studies, and no inflammatory or pyrogenic reactions were observed. When the LP test was used, experiments revealed that the original lot of met-hGH was contaminated with endotoxin which had not been detected in the LAL or rabbit pyrogen tests. Lyophilization in glycine-phosphate buffer had resulted in a 10- to 20-fold reduction of endotoxin reactivity in the LAL test and the U.S. Pharmacopeial Convention rabbit pyrogen test. These data provide a probable explanation for the negative result from the LAL and rabbit pyrogen test in the initial lot of met-hGH which induced acute-phase reactions. In addition, these studies demonstrate that the release of LP from human cells is a reliable indicator of the presence of materials that are pyrogenic for humans.